The following proposal addresses the need for defining an etiology of primary progressive multiple sclerosis (PPMS), a severe demyelinating disease of unknown cause, which affects the brain and spinal cord. PPMS is the most acute and severe manifestation of MS, affecting about 40,000 individuals in the U.S. Disease-modifying treatments, effective for relapsing-remitting MS, have been ineffective for PPMS. A number of central nervous system viral infections can lead to demyelination, including distemper in dogs and measles (SSPE) and influenza in humans. Viruses have long been suspected as causative agents in multiple sclerosis based on geographic patterns of disease, isolated outbreaks, and migration studies. Novel unculturable viruses that cause human disease continue to be discovered using molecular techniques including hepatitis C (1991), corona virus NL63 (2004), bocavirus (2005), and rhinovirus C group (2007). New human polyoma and arenaviruses (2008) were recently identified by deep sequencing - the technique proposed here for the present investigation of PPMS brain tissue. Preliminary data was developed by deep sequencing of RNA from MS plaques. These initial sequencing data have led to an intriguing candidate virus: GBV-C, a member of the Flaviviridae family never recovered before from a human brain. GBV-C has some interesting epidemiologic and clinical features that warrant additional study of the role of this virus in PPMS. The present proposal seeks to apply improved sequencing techniques to PPMS brain specimens in comparison to normal and other neurologic disease controls (N = 15 each group). Sequencing data will be analyzed for virus-like sequences. Data from control brain specimens will be made available to the scientific community. The preliminary and newly acquired data will be used to design primers and screen for GBV-C, and any additional virus candidates that may emerge, in banked MS specimens, including tissue and CSF. This approach is unbiased and data driven - there are no preconceptions about which viruses should be tested for. This project uses a new tool - deep sequencing - to examine specimens collected from two U.S. brain banks. It has already provided a new lead into the pathogenesis of MS (i.e. the candidate virus listed above). Amplification of any novel or previously unsuspected virus from MS brain, significantly different than controls, would likely spur intensive research. A proven pathogenic virus, even if only in a small proportion of MS patients, could lead to high impact blood and CSF tests for this virus. It is also conceivable that deep sequencing itself could be developed to the point that blood, CSF, or diseased brain tissue could be examined for potential pathogens in living patients. Eventually, specific and potentially highly effective antiviral treatment could be developed in at least a subset MS patients. Finally, if this work is successful it may be possible to develop one or more vaccines to prevent the onset of MS.